Fig. 5: Differential post-translational modifications alter DRP1 localisation. | Cell Death & Disease

Fig. 5: Differential post-translational modifications alter DRP1 localisation.

From: ISGylation of DRP1 closely balances other post-translational modifications to mediate mitochondrial fission

Fig. 5

A Cytosolic and mitochondrial fractions obtained from semi-permeabilised cells, transfected with indicated constructs, were immunoblotted using antibody against DRP1. GAPDH and VDAC1 served as controls for cytosolic and mitochondrial fractions, respectively. Note lower levels of DRP1 were observed in samples with CoV2 PLpro WT. Unmodified DRP1, ← and likely to be post-translationally modified forms of DRP1. Note reduced DRP1 levels in input (exposure 1), mitochondrial (exposure 2) and cytosolic (exposure 3) fractions in CoV2 PLpro WT samples. (B) Total RNA isolated from A549 cells transfected with the indicated GFP-tagged constructs was subjected to qRT-PCR using primers against GAPDH (control) and DRP1. Samples were present in triplicate. 2-ΔΔCt values were plotted. Graph shows results from 3 independent experiments. ns, not significant (p > 0.1) using unpaired 2-tailed Student’s t-test. Error bars, ±SEM. C Cytosolic and mitochondrial fractions obtained from semi-permeabilised cells were analysed for co-IP between DRP1 and ISG15. Note that cytosolic DRP1 pool is primarily ISGylated. VINCULIN and VDAC1 served as controls for cytosolic and mitochondrial fractions, respectively. D Cells fractionated as in panel C were probed for co-IP between DRP1and Ub. Note ubiquitylated DRP1 present in both the fractions. VDAC1 and VINCULIN levels in the total lysates served as loading controls. E Cells transfected with the indicated GFP-tagged constructs, fractionated as in panel C were divided into two. One part was immunoprecipitated with anti-ISG15 antibody and immunoblotted against DRP1. Note lower levels of ISGylated DRP1 in samples with CoV2 PLpro WT than the mutant. VINCULIN and VDAC1 served as controls. F Second part generated in panel E was analysed for co-IP between DRP1 and Ub. VDAC1 and VINCULIN levels confirm fractionation efficiency. G Cytosolic and mitochondrial fractions obtained from semi-permeabilised cells were analysed for TRIM25. VINCULIN and VDAC1 served as controls.

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